PROJECT SUMMARY Significance: Numerous RNA modifying enzymes are critical for human health but the locations and stoichiometry of most RNA modifications are currently unknown. This is primarily due to the lack of high- throughput methods to detect the majority of modified nucleosides. Dihydrouridine (D) is an intriguing and understudied RNA modification that profoundly affects RNA backbone conformation, and, as a result, RNA metabolism. The RNA modifying enzymes that install dihydrouridine (D), called dihydrouridine synthases (DUS), are overexpressed and predictive of worse patient outcomes in lung, kidney and bladder cancers. The association of elevated DUS expression with poorer prognosis is consistent with a pathophysiological effect of increased dihydrouridine at some sites, which could include increased stoichiometry of D at known target sites or modification of new sites, or both. The specific RNA targets of DUS enzymes in cancer cells are unknown. Approach: Here, we propose to develop technology for comprehensive genomic analysis of dihydrouridine (D) (Aim 1) and use it to determine how the D landscape is altered in kidney cancer cells (Aim 2). Our approach exploits unique chemical features of dihydrouridine to derivatize D nucleotides, enrich for D containing RNA, and determine the locations of D with single-nucleotide resolution. Our preliminary data establish selectivity for D and the ability to generate precise modification-dependent blocks to reverse transcriptase, which we will analyze by Illumina sequencing. The probability of technical success is high: our laboratory is a technological pioneer in the discovery of RNA modification sites by developing experimental and computational methods to map the locations and quantify the relative abundance of novel mRNA modifications on a transcriptome-wide scale with single- nucleotide resolution. This proposal leverages these accomplishments to develop new methods to study the dihydrouridine landscape in human cells and reveal its dysregulation in cancer. The goal of this exploratory project is to discover the specific RNA targets of the DUS1L and DUS3L enzymes whose overexpression predicts poor prognosis in kidney cancer. Notably, DUS1L and DUS3L associate with polyadenylated RNA in cells and so the D landscape is likely to be complex and include sites in mRNA that are currently undiscovered. Aim 1 will develop high-throughput methods to define comprehensive targets of human DUS (1A) and medium-throughput methods to measure absolute dihydrouridine stoichiometry at hundreds of sites in parallel (1B). In Aim 2, we will use these methods to discover the targets of DUS1L and DUS3L and determine how the locations and stoichiometry of D modifications are altered in clear cell renal cell carcinoma cell lines that express high levels of DUS1L and DUS3L compared with matched DUS knockout cells (2A) and with non-transformed renal proximal tubule epithelial cells (2B). We anticipate that the methods developed here will be broadly useful for charting alterations in the epitranscriptome during initiation and progression of multiple malignancies where altered expression of dihydrouridine ?writers? is implicated in disease.